1. Field of the Invention
The present invention relates to an apparatus for analyzing an extremely small quantity of a wide variety of applications such as protein, nucleic acid or the like, at high speed and in high resolution. More particularly, it relates to a microchip electrophoresis apparatus employing a microchip.
2. Description of the Prior Art
A capillary electrophoresis apparatus is generally employed for analyzing an extremely small quantity of protein or nucleic acid. The capillary electrophoresis apparatus charges a glass capillary with an inner diameter of not more than 100 .mu.m with a migration buffer, introduces a sample into an end thereof, and subsequently applies a high voltage across the glass capillary to separate molecules based on the differences in charge-to-size ratio. The capillary has a large surface area relative to its volume, (i.e., high cooling efficiency). In this way, a high voltage can be applied to the capillary for analyzing an extremely small quantity of sample, such as DNA, at a high speed in high resolution.
However, the capillary has a small outer diameter of about 100 to 400 .mu.m, and is fragile even though it is usually protected by a polyimide coating. Therefore, the user must be extremely careful during the process of exchanging it Furthermore, the accurately measured injection of sample into a capillary is difficult, and on-capillary reaction schemes usually require junctions which are difficult or tedious to make without introducing extra volume. These have led to the proposal of a capillary electrophoretic chip (referred to as a microchip in this application) formed by connecting two substrates to each other, as described in Anal. Chim. Acta 283 (1993) pp. 361-366 by D. J. Harrison et al. FIGS. 1A to 1D show an exemplary microchip. This microchip comprises a pair of transparent substrates 1 and 2. Substrate 2 is provided with migration capillary grooves 4 and 5, which are formed by etching, which intersect each other, while substrate 1 is provided with through holes 3 in positions corresponding to both ends of grooves 4 and 5.
When employing this microchip, substrate 1 and 2 are superimposed over one another as shown in FIGS. 1 C and 1 D, so that an electrolyte or an electrophoretic buffer solution (hereinafter refer to as "buffer solution") may be injected into grooves 4 and 5 from any through hole 3. Following this, thereafter a sample is injected from through hole 3(S), which is located at one end of shorter groove 4, and a high voltage is then applied between through holes 3(S) and 3(W), which are located at both ends of groove 4, for a prescribed time. In this way, the sample is dispersed in groove 4.
Following this, a separation voltage for electrophoretic separation is applied between through holes 3(B) and 3(D), which are located at both ends of longer groove 5. Thus, the sample which is present on intersection 6, between grooves 4 and 5, is electrophoresed in groove 5. A detector, such as an ultraviolet-visible spectrophotometer, a fluorophotometer, or an electrochemical detector, is located in a position relative to groove 5 in order to detect the separated component
It has been demonstrated that such electrophoresis with a microchip is capable of high-speed separation and microanalysis in miniaturized system. If instrumentation of the electrophoresis progresses, there is the potential of attaining a completely new and unique analyzer.
In the aforementioned technique employing the microchip, the buffer solution is manually charged in grooves 4 and 5 from any through hole 3. The sample is also manually injected into through hole 3(S). Through hole 3, which is used for the buffer solution, serves as a reservoir for the buffer solution, and through hole 3(S), for injecting the sample, acts as a sample container. Pre-analytical operations entail manually feeding the buffer solution into any through hole 3 with a syringe or similar instrument, and injecting the sample into through hole 3(S), which is provided on one end of groove 4, with another syringe.
FIG. 2 shows an exemplary electrophoresis apparatus employing a microchip 10.
X-Y stage 12 is placed on optical bench 14 as a mechanism for moving microchip 10 in a horizontal plane. Microchip 10 is attached to X-Y stage 12 and manually moved in the horizontal plane along directions X and Y. A laser induced fluorescence detector, which excites a given sample by means of laser beam for detecting it through its fluorescence emission, optically detects the sample separated by electrophoresis in a migration passage. The laser beam from laser unit 16 is passed into a confocal microscope 18 and reflected with a dichroic mirror to an objective, which focused the sample injected into microchip 10. The fluorescence generated from the sample is collected by the same objective, passed through the dichroic mirror, filtered by a bandpass filter and focesed on a pinhole followed by photomultiplier 20 detection. Binocular 22 adjusts the optical axis of the irradiating and condensing part 18. Laser unit 16, confocal microscope 18 and binocular 22 are also arranged on optical bench 14. Numeral 24 denotes a laser power source, while numeral 26 denotes a high-voltage power source for photomultiplier 20. Amplifier 28 amplifies an optical signal detected by photomultiplier 20, and A-D converter 30 converts the amplified signal to a digital signal, so that CPU 32 is able to retrieve this digital signal.
High-voltage power sources 34 and 36 are provided for applying a sample introduction voltage to introduce the sample, which is injected into microchip 10, into a separation passage and a separation voltage for electrophoretically separating the sample respectively. High-voltage power sources 34 and 36 apply the voltages to microchip 10 through relay control system 38. CPU 32 serves as a control unit, switching the sample introduction voltage and the separation voltage through relay control system 38, and collecting data from A-D converter 30 prior to processing it CPU 32 is connected to external personal computer 39 for transmittng and receiving the data.
In the electrophoresis apparatus shown in FIG. 2, the buffer solution must be introduced into the passage of microchip 1O with a syringe or similar instrument, and the sample of several .mu.l must also be introduced into sample reservoir S, by similar means before microchip 10 is set on X-Y stage 12. The laser beam must be focused on a point of the separation passage in order to detect the electrophoretically separated sample. The optical alignment of the system is made visually using a three-axis translation stage with respect to a fixed microscope.
This electrophoresis apparatus requires a preliminary operation of the filling up of microchip 10 with the buffer solution and subsequent positioning of the sample on sample reservoir S. Additionally, the optical axis of the laser beam must be adjusted to the separation passage of microchip 10 which requires the strict alignment of microchip 10 with the detector in order to efficiently condense the fluorescence. Therefore, large-scale devices such as binocular 22, optical bench 14, an optical axis adjusting mechanism, general-purpose high-voltage power sources 34 and 36, laser unit 16 and power source 24 are required, although microchip 10, itself, is relatively small (something in the region of 20 mm by 40 mm,), and is employed for microanalysis which hardly consumes a reagent